The goal of this renewal application is to define the largely unexplored relationship between the ATM/ATR/Chk1 DNA-damage checkpoint machinery and the Src/Ras/Raf/MEK/ERK survival signaling pathway in multiple myeloma (MM) cells, and to exploit this knowledge therapeutically. The previous project stemmed from the observation that the multi-kinase and Chk1 inhibitor UCN-01 triggered MEK/ERK activation in MM cells, and that pharmacologic interruption of this pathway strikingly induced apoptosis in malignant cells, including primary CD138+, but not normal cells. During the preceding period, important new insights emerged, including the observations that a) disruption of Chk1 by UCN-01 activates MEK1/2/ERK1/2 through a Ras- dependent mechanism that limits lethality; b) these findings may represent a generalized phenomenon involving novel and more specific Chk1 inhibitors as well as new agents acting upstream of Chk1 (i.e., ATM/ATR inhibitors); c) interrupting the MEK/ERK pathway at various levels, e.g., by new, clinically relevant MEK1/2 inhibitors as well as antagonists of upstream signaling targets (e.g., Src and Ras) dramatically potentiates checkpoint abrogator lethality; d) the lethality of these regimens is associated with marked potentiation of DNA damage and induction of Bim-dependent apoptosis; e) this approach is highly lethal toward non-cycling G0G1 MM cells and circumvents Mcl-1-related resistance; and e) this strategy is effective in vivo. Collectively, our findings argue that in MM, activation of the Ras/Raf/MEK/ERK pathway represents a critical cytoprotective response to Chk1/ATM/ATR inhibitors, and that blockade of the former pathway leads to pronounced activity against MM cells both in vitro and in vivo. The specific aims of this proposal are to 1) validate, through genetic means, the Chk1 and MEK/ERK pathways as targets for regimens combining new generation Chk1 and ATM/ATR inhibitors with MEK/ERK pathway antagonists in MM, and investigate mechanisms responsible for synergistic interactions, focusing on Bim upregulation, DNA damage induction, and cdc2 activation; 2) test the activity and selectivity of novel regimens toward primary CD138+ MM cells, and elucidate mechanisms responsible for the striking susceptibility of non-cycling G0G1 and primary MM cells to this strategy; 3) define the basis by which these regimens circumvent cytoprotection by Mcl-1, a critical MM survival factor, and use genetic and pharmacologic strategies to determine whether disabling of Bcl-2/Bcl-xL improves therapeutic activity further; and 4) determine whether regimens combining second-generation Chk1 and ATM/ATR inhibitors with MEK/ERK pathway antagonists overcome conventional, novel (e.g., stromal-cell related), and bortezomib resistance, emphasizing in vivo flank and systemic xenograft MM model systems. These studies will establish a new treatment paradigm for Chk1 inhibitors and lay the foundation for one or more novel Phase I trials in refractory MM in which emerging inhibitors of the ATM/ATR/Chk1 checkpoint machinery and the MEK/ERK survival pathway are rationally combined to trigger MM cell death.